Cytosine methylation pattern are established early in development. Although several DNA methyltransferases have been identified it remains unknown how specific genomic methylation patterns are generated that allow tissue specific gene expression. We examined the possibility that Lsh controls not only cytosine methylation at repetitive sequences, but may be also involved in the control of DNA methylation at unique promoter regions. To investigate how genome-wide DNA methylation patterns are established in mice and how they may specifically depend on Lsh, we generated a comprehensive genomic map of cytosine methylation in cells derived from mice with a targeted deletion of Lsh. We performed a MeDIP assay (immunoprecipitation of methylated DNA using anti-methyl-cytidine antibody) comparing DNA derived from wild type murine embryonal fibroblast cell lines (MEFs) with Lsh-/- MEFs. The precipitated DNA was hybridized to the MM8 tiling whole mouse genomic array which covers a region of about 3 billion bps excluding repeat masked regions at a resolution of a 100 bp. In addition, a histone 3 lysine 4 tri-methylation (H3K4me3) chromatin map was generated using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) and genome-wide gene expression was evaluated using cDNA microarrays. Total MeDIP enrichment of the genomic array was of similar magnitude comparing WT to the Lsh-/- sample, indicating that there was not a major net loss of cytosine methylation at unique sequences. Chromosomal maps based on mean methylation enrichment values at 50 kb windows revealed specific sites that were either hypo- or hypo-methylated in the absence of Lsh. For the whole genome about 6.4% or 8.6% was distinctively differentially methylated (hypo- or hypermethylated) in the absence of Lsh. Contiguous regions for cytosine methylation changes extended up to 2Mb suggesting that discrete chromosomal domains share a common epigenetic pathway, controlled by Lsh. Cytosine methylation pattern at protein coding genes revealed differences at the 5-end but not at the 3-ends of genes comparing wild type and Lsh-/- samples. A subset of genes containing CpG islands exhibited cytosine hypermethylation at their 5-ends. For the majority of genes the hypermethylation was associated with an increase in H3K4me3 enrichment, but a small subset showed a loss of H3k4me3. In order to gain further insight into how changes in cytosine methylation may be linked to gene expression, we analyzed the subset of genes that were differentially expressed more than two-fold. Whereas up-regulated genes had H3K4me3 increases in conjunction with DNA methylation changes, down-regulated genes displayed unchanged or reduced H3K4me3 or in the immediate TSS region in Lsh-/- MEFs. This suggests that the combination of both epigenetic marks (cytosine methylation and H3K4me3) determines the outcome of gene expression and, in addition, that DNA methylation increases in Lsh-/- MEFs can be linked to up- or down-regulation. Gene ontology analysis of differentially expressed genes in Lsh-/- MEFs showed an enrichment for genes involved in angiogenesis, lung development, skeletal system development, embryonic morphogenesis, and organ development, thus pointing to a role of Lsh in murine development. Taken together, Lsh controls the normal distribution of cytosine methylation at chromosomal domains and at promoter regions. Thus Lsh is a genome wide epigenetic regulator at non-repetitive sequences and plays a unique role in gene expression during embryonic development. Elucidating the basic mechanisms of epigenetics provide fundamental insights into biologic processes of cellular differentiation, cellular transformation and nuclear reprogramming.